John S. Beattie

A multiplexed protein microarray for the simultaneous serodiagnosis of human immunodeficiency virus/hepatitis C virus infection and typing of whole blood**

All donor blood samples must be tested pretransfusion to determine the donor blood type. Standard testing protocols require that assays be performed for important bloodborne pathogens such as hepatitis C, syphilis, hepatitis B, and human immunodeficiency virus. We have demonstrated proof of the concept that a protein microarray can type whole blood and detect antibody to significant pathogens simultaneously from the same donor blood sample. The data collected demonstrate the ability of the array to accurately type blood samples while also detecting the presence of antibodies against both human immunodeficiency virus and hepatitis C virus. In conclusion, we have successfully developed a platform capable of typing human whole blood samples, while at the same time testing for the presence of antibodies specific for human immunodeficiency virus/hepatitis C virus. The major benefits of this system are its amenability to expansion with additional assays, for example, rhesus typing and syphilis and/or hepatitis B virus detection, and also the adaptability of the assay to higher-throughput analysis, currently 16 individual samples per slide, but readily expandable to a 96-well format.

PDQ Wizard: automated prioritization and characterization of gene and protein lists using biomedical literature

SUMMARY PDQ Wizard automates the process of interrogating biomedical references using large lists of genes, proteins or free text. Using the principle of linkage through co-citation biologists can mine PubMed with these proteins or genes to identify relationships within a biological field of interest. In addition, PDQ Wizard provides novel features to define more specific relationships, highlight key publications describing those activities and relationships, and enhance protein queries. PDQ Wizard also outputs a metric that can be used for prioritization of genes and proteins for further research. AVAILABILITY PDQ Wizard is freely available from http://www.gti.ed.ac.uk/pdqwizard/.

GPX-Macrophage Expression Atlas: A database for expression profiles of macrophages challenged with a variety of pro-inflammatory, anti-inflammatory, benign and pathogen insults

BackgroundMacrophages play an integral role in the host immune system, bridging innate and adaptive immunity. As such, they are finely attuned to extracellular and intracellular stimuli and respond by rapidly initiating multiple signalling cascades with diverse effector functions. The macrophage cell is therefore an experimentally and clinically amenable biological system for the mapping of biological pathways. The goal of the macrophage expression atlas is to systematically investigate the pathway biology and interaction network of macrophages challenged with a variety of insults, in particular via infection and activation with key inflammatory mediators. As an important first step towards this we present a single searchable database resource containing high-throughput macrophage gene expression studies.DescriptionThe GPX Macrophage Expression Atlas (GPX-MEA) is an online resource for gene expression based studies of a range of macrophage cell types following treatment with pathogens and immune modulators. GPX-MEA follows the MIAME standard and includes an objective quality score with each experiment. It places special emphasis on rigorously capturing the experimental design and enables the searching of expression data from different microarray experiments. Studies may be queried on the basis of experimental parameters, sample information and quality assessment score. The ability to compare the expression values of individual genes across multiple experiments is provided. In addition, the database offers access to experimental annotation and analysis files and includes experiments and raw data previously unavailable to the research community.ConclusionGPX-MEA is the first example of a quality scored gene expression database focussed on a macrophage cellular system that allows efficient identification of transcriptional patterns. The resource will provide novel insights into the phenotypic response of macrophages to a variety of benign, inflammatory, and pathogen insults. GPX-MEA is available through the GPX website at http://www.gti.ed.ac.uk/GPX.

Molecular recognition with DNA nanoswitches: effects of single base mutations on structure.

This paper investigates the properties of a simple DNA-based nanodevice capable of detecting single base mutations in unlabeled nucleic acid target sequences. Detection is achieved by a two-stage process combining first complementary-base hybridization of a target and then a conformational change as molecular recognition criteria. A probe molecule is constructed from a single DNA strand designed to adopt a partial cruciform structure with a pair of exposed (unhybridized) strands. Upon target binding, a switchable cruciform construct (similar to a Holliday junction) is formed which can adopt open and closed junction conformations. Switching between these forms occurs by junction folding in the presence of divalent ions. It has been shown from the steady-state fluorescence of judiciously labeled constructs that there are differences between the fluorescence resonance energy transfer (FRET) efficiencies of closed forms, dependent on the target sequence near the branch point, where the arms of the cruciform cross. This difference in FRET efficiency is attributed to structural variations between these folded junctions with their different branch point sequences arising from the single base mutations. This provides a robust means for the discrimination of single nucleotide mismatches in a specific region of the target. In this paper, these structural differences are analyzed by fitting observed time-resolved donor fluorescence decay data to a Gaussian distribution of donor-acceptor separations. This shows the closest mean separation (approximately 40 A) for the perfectly matched case, whereas larger separations (up to 50 A) are found for the single point mutations. These differences therefore indicate a structural basis for the observed FRET differences in the closed configuration which underpins the operation of these devices as biosensors capable of resolving single base mutations.

Electrochemical control of a DNA Holliday Junction nanoswitch by Mg2+ ions.

The molecular conformation of a synthetic branched, 4-way DNA Holliday junction (HJ) was electrochemically switched between the open and closed (stacked) conformers. Switching was achieved by electrochemically induced quantitative release of Mg(2+) ions from the oxidised poly(N-methylpyrrole) film (PPy), which contained polyacrylate as an immobile counter anion and Mg(2+) ions as charge compensating mobile cations. This increase in the Mg(2+) concentration screened the electrostatic repulsion between the widely separated arms in the open HJ configuration, inducing switching to the closed conformation. Upon electrochemical reduction of PPy, entrapment of Mg(2+) ions back into the PPy film induced the reverse HJ switching from the closed to open state. The conformational transition was monitored using fluorescence resonance energy transfer (FRET) between donor and acceptor dyes each located at the terminus of one of the arms. The demonstrated electrochemical control of the conformation of the used probe-target HJ complex, previously reported as a highly sequence specific nanodevice for detecting of unlabelled target [Buck, A.H., Campbell, C.J., Dickinson, P., Mountford, C.P., Stoquert, H.C., Terry, J.G., Evans, S.A.G., Keane, L., Su, T.J., Mount, A.R., Walton, A.J., Beattie, J.S., Crain, J., Ghazal, P., 2007. Anal. Chem., 79, 4724-4728], allows the development of electronically addressable DNA nanodevices and label-free gene detection assays.

A DNA nanoswitch incorporating the fluorescent base analogue 2-aminopurine detects single nucleotide mismatches in unlabelled targets.

DNA nanoswitches can be designed to detect unlabelled nucleic acid targets and have been shown to discriminate between targets which differ in the identity of only one base. This paper demonstrates that the fluorescent base analogue 2-aminopurine (AP) can be used to discriminate between nanoswitches with and without targets and to discriminate between matched and mismatched targets. In particular, we have used both steady-state and time-resolved fluorescence spectroscopy to determine differences in AP environment at the branchpoint of nanoswitches assembled using complementary targets and targets which incorporate single base mismatches.

Evaluation of a Protein Microarray Method for Immuno‐Typing Erythrocytes in Whole Blood

Abstract All donor blood samples must be tested pre‐transfusion to determine the blood type of donor erythrocytes, based on the ABO typing system. Current methods of testing are well characterised, but require a number of processing steps prior to analysis. In addition, standard testing protocols require additional assays such as hepatitis C and HIV testing be performed separately. We describe and evaluate a protein microarray platform for ABO blood typing that has the potential to be a simple reliable high throughput method, with the added capability for the integration of other important pre‐transfusion tests. Sixty seven donor blood samples were incubated on microarrays printed with multiple spotted replicates of blood type antigen specific antibodies. We utilised a hold‐out cross validation approach, combined with Receiver Operator Characteristic (ROC) curves to define thresholds within which a sample could be defined as being of a particular blood type. The threshold values from the ROC curve analysis demonstrated an excellent ability to accurately separate samples based on ABO blood type. The results obtained when the thresholds from the training sets were applied to test sets were also very encouraging, with misclassified samples being present in only 2 of the training sets and a mean classification error of 4.28%. When the mean thresholds were applied to the 67 donor samples, 95.5% were correctly blood typed (64 of 67 samples). We have demonstrated the ability of our protein microarray platform to successfully and accurately type human whole blood samples. We believe that this flexible platform provides a strong basis for an integrated approach for combined blood typing and pathogen testing in human whole blood.

Time-Resolved FRET and FLIM of Four-way DNA Junctions

Conformational transitions in a 4-way DNA junction when titrated with ionic solutions are studied using time-resolved fluorescence resonance energy transfer. Parameters characterising the transition in terms of critical ion concentration (c1/2) and the Hill coefficient for ion binding are obtained by fitting a simple two-state model using steady-state spectra. Data obtained from a fluorescence lifetime plate reader and analysed by fitting a single exponential to donor fluorescence lifetime decays are shown to be in good agreement with the parameters obtained from steady-state measurements. Fluorescence lifetimes, however, offer advantages, particularly in being independent of fluorophore concentration, output intensity, inhomogeneity in the excitation source and output wavelength. We demonstrate preliminary FRET-FLIM images of DNA junction solutions obtained using a picosecond gated CCD which are in agreement with results from a fluorescence lifetime plate reader. The results suggest that time-resolved FRET-FLIM is sensitive to subtle structural changes and may be useful in assays based on 4-way DNA junctions.

Interdisciplinary integrated research teams in an academic environment

This paper presents a discussion on the extent to which industrial R&D management techniques can be applied in an academic research environment. Working in highly integrated interdisciplinary teams has been shown to provide significant benefits in industrial R&D, yet organisational forms, reward systems and the culture of the academic environment remain a barrier to team working. A group of four research centres at the University of Edinburgh have trialled a new integrated team approach similar to the use of high-performance teams in industrial product development. This has proved a highly effective method of carrying out novel interfacial research, allows groups to address more substantial research questions and generates a truly interdisciplinary research capability for further work. Using this project as a case study has provided an insight into the value of this approach and the specific issues to be considered.

Positional characteristics of fluorophores influencing signal output of a DNA nanoswitch

The Holliday junction (HJ) structure, consisting of four DNA double helices with a central branch point, is capable of switching between conformational states upon ion binding. The HJ nanoswitch described here comprises a long, dual labeled cloverleaf oligonucleotide and a short, unlabeled oligonucleotide. Fluorescent labeling with donor and acceptor dyes placed on the HJ arms of the cloverleaf strand allows the ion induced conformational switch to be detected optically using fluorescence resonance energy transfer (FRET). The influence of donor and acceptor dye location on the detection of conformational switching has been investigated using two distinct HJ structures. In addition, the effect of increasing HJ arm length in order to increase donor and acceptor dye separation has been evaluated. We report that a preferential HJ nanoswitch structure can be determined, capable of efficient detection of ion induced conformational switching